Showing papers on "Alkaline phosphatase published in 1984"

Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).

Abstract: A murine monoclonal antibody specific for calf intestinal alkaline phosphatase has been prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure--the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) method--gives excellent immunocytochemical labeling of tissue sections and cell smears, comparable in clarity and intensity to that achieved with immunoperoxidase labeling. If the enzyme label is developed with a naphthol salt as a coupling agent and Fast Red or hexazotized new fuchsin as a capture agent, a vivid red reaction product is obtained which is very easily detected by the human eye. For this reason the APAAP technique was found particularly suitable for labeling cell smears (for both cytoplasmic and surface-membrane antigens) and for detecting low numbers of antigen-bearing cells in a specimen (e.g., carcinoma cells in a malignant effusion). It was found possible to enhance the intensity of the APAAP labeling reaction substantially by repeating the second and third incubation steps (i.e., the unlabelled "bridge" antibody and APAAP complexes). The APAAP technique was superior to immunoperoxidase labeling for staining tissues rich in endogenous peroxidase, and could be used in conjunction with immunoperoxidase methods for double immunoenzymatic staining. The method was also applicable to the detection of antigenic molecules following their electrophoretic transfer from SDS-polyacrylamide gels to nitrocellulose sheets ("immunoblotting").

Two new methods for separating and quantifying bone and liver alkaline phosphatase isoenzymes in plasma.

Abstract: We describe two new methods for the separation and quantification of the bone and liver isoenzymes of alkaline phosphatase (EC 3.1.3.1) in plasma. In the first, we use wheat-germ lectin to precipitate the bone isoenzyme. About 80% of this, but minimal liver isoenzyme, is precipitated. The activity of the bone isoenzyme is calculated from measuring the alkaline phosphatase activity in the precipitate, that of liver alkaline phosphatase by subtracting the activity of the bone isoenzyme from total alkaline phosphatase activity. The liver fraction will also contain biliary, intestinal, and placental alkaline phosphatase if these are present in the original plasma, but correction for such activity is readily made. In the second method, samples are separated on cellulose acetate membranes that, before electrophoresis, have been soaked in buffer containing wheat-germ lectin. The bone isoenzyme is retarded and clearly separated from the liver fraction, allowing these isoenzymes to be quantified by densitometry. Both methods are rapid, reproducible, and suitable for use in the diagnostic laboratory.

Effect of growth hormone on osteoblasts and demonstration of somatomedin-C/IGF I in bone organ culture

Abstract: Bone organ culture makes it possible to observe the direct influence of hormones on bone cells. We studied the effect of growth hormone in vitro on embryonal rat tibiae during culture for 7 days, functionally by measuring the levels of alkaline phosphatase in the culture medium, and morphologically by means of semi-thin sections and electron microscopic examination. Since growth hormone (GH) is supposed to exert an indirect effect on bone cells, somatomedin-C/insulin-like growth factor I (SM-C/IGF I) as a possible mediator was also measured radioimmunologically in the culture medium. In the controls alkaline phosphatase levels showed a continuous increase up to the 7th day which was significantly higher in the presence of GH. There was also a significantly enhanced increase of SM-C/IGF I in the presence of GH during culture in comparison to the controls. Evidently IGF I is produced locally in bone and mediates the effect of GH on bone formation.

Characterization of cells isolated and cultured from human bone.

Abstract: Cells isolated from samples of human iliac crest and human femoral heads by collagenase digestion have been successfully cultured in Fitton-Jackson modified BGJb culture medium supplemented with penicillin (100 units/ml), streptomycin (100 micrograms/ml), and fetal calf serum (10%). Although only a low proportion of the cells survived the initial plating (less than 1%), cells established in culture were readily passaged. Examination of cells obtained at intervals during the collagenase digestion showed that the percentage of cells that attached increased with time of digestion. Rapid sample preparation of rat bone did not substantially increase the number of cells attaching. Thus, it seems unlikely that the low survival was due to loss of viability during sample transportation and preparation. Of several media tested BGJb supplemented with 10% fetal calf serum supported the best growth. Population doubling time averaged 104 hr. Cultured human bone cells were assayed for alkaline phosphatase activity using the azo dye method with naphthol ASTR phosphate as the substrate. A portion of the cells (19%) demonstrated high activity in all cultures examined regardless of the passage number of the culture. Autoradiography of cells exposed to [3H]thymidine showed incorporation of the label into both alkaline phosphate-positive and -negative cells. The stimulation of cell proliferation by growth factors was studied by determining the incorporation of [3H]thymidine into DNA. The specific skeletal growth factor from human bone stimulated cell proliferation several-fold with a half-maximal effect at 5 micrograms/ml. Insulin, epidermal growth factor, and a crude preparation of somatomedin C also stimulated cell proliferation.

Heat-labile alkaline phosphatase from Antarctic bacteria: Rapid 5′ end-labeling of nucleic acids

Abstract: A heat-labile alkaline phosphatase has been purified to near homogeneity from HK47, a bacterial strain isolated from Antarctic seawater. The active form of the enzyme has a molecular weight of 68,000 and is uniquely monomeric. The optimal temperature for the enzymatic activity is 25°C. Complete and irreversible thermal inactivation of the enzyme occurs in 10 min at 55°C. By using this heat-labile enzyme for dephosphorylation followed by a 10-min heat treatment, rapid end-labeling of nucleic acids by T4 polynucleotide kinase has been achieved.

Isolation of oval cells by centrifugal elutriation and comparison with other cell types purified from normal and preneoplastic livers.

Abstract: Oval cells and biliary epithelial cells were isolated from livers of rats fed a choline-deficient diet containing 0.1% ethionine and from normal rat livers, respectively. Nonparenchymal cell suspensions prepared from these livers by collagenase perfusion followed by digestion of undissociated tissue with 0.1% collagenase, 0.1% Pronase, and 0.004% DNase I were separated into six fractions by centrifugal elutriation. Cells in each fraction were characterized histochemically for gamma-glutamyl transpeptidase, peroxidase, alkaline phosphatase, and glucose-6-phosphatase activities, and for albumin and alpha-fetoprotein by immunocytochemical methods. Cells from Fraction 5 of the elutriation procedure had various features predicted for oval cells and were selected for further studies. The cell yield in this fraction, from each preneoplastic liver, was 5.7 X 10(7) cells, 93 +/- 2% of which were gamma-glutamyl transpeptidase positive, 6 +/- 1% peroxidase positive, 61% albumin positive, and 29% alpha-fetoprotein positive. Cells in this fraction have a median diameter of 13.1 micron and are diploid and cycling. The majority of these cells has morphological features characteristic of biliary epithelial cells, although some cells display features intermediate between duct cells and hepatocytes. Nucleic acid hybridization using specific probes revealed that these cells contain albumin and alpha-fetoprotein messenger RNAs, while hepatocytes from normal and preneoplastic liver contain only albumin messenger RNA. Biliary cells obtained from normal livers do not contain albumin messenger RNA. The large-scale purification and characterization of cell populations from preneoplastic livers is an important step in elucidating the cellular derivation of liver tumors.

Effects of acute changes of bile acid pool composition on biliary lipid secretion

Abstract: To elucidate the mechanism responsible for the bile acid-induced changes of biliary lipid secretion, we evaluated bile flow and biliary output of bile acids, cholesterol, phospholipids, and alkaline phosphatase activity in seven cholecystectomized subjects with a balloon occludable T-tube during two experimental periods: (a) depletion of the endogenous bile acid pool and (b) replacement of the pool by means of duodenal infusion with individual bile acids, such as deoxycholic (DCA), chenodeoxycholic (CDCA), cholic (CA), and ursodeoxycholic (UDCA) acids. Bile flow, cholesterol, and phospholipid output were linearly related to bile acid secretion in all experimental periods. During the replacement periods, the amount of cholesterol and phospholipids coupled to bile acids was significantly different (at 1% level at least) for each individual bile acid secreted; it was the highest during DCA secretion (slope value: 0.209 for cholesterol and 0.434 for phospholipids) followed, in the order, by CDCA (0.078 and 1.794), CA (0.044 and 0.127), and UDCA (0.030 and 0.122). The phospholipid to cholesterol ratio was higher during secretion of CA and UDCA as compared with DCA and CDCA. The secretion of CA seemed to stimulate a greater bile flow than the other bile acids did. The infusion of all bile acids, except UDCA, induced an increase of biliary alkaline phosphatase activity as compared with the values of the depletion period. The mean highest increase (13-fold the pretreatment value) was observed during DCA secretion followed by CDCA (fivefold) and CA (1.5-fold). These results would suggest that the physical chemical properties, namely the lipid-solubilizing capacity, of bile acids could directly contribute to the regulation of biliary lipid secretion. The observed changes in biliary alkaline phosphatase activity lend support to the view that bile acid-induced lipid secretion may be, at least in part, contributed by membrane solubilization.

A phosphotyrosyl-protein phosphatase activity associated with acid phosphatase from human prostate gland

Abstract: Using [32P]P-Tyr-IgG and [32P]P-Tyr-casein phosphorylated by pp60v-src as substrates, studies on the phosphotyrosyl-protein phosphatase activity in human prostate gland indicate that it is associated with prostatic acid phosphatase. Evidence to support this conclusion include the following: (a) these two enzymatic activities co-purify to apparent homogeneity; (b) they co-migrated on polyacrylamide gel electrophoresis, ion-exchange and gel filtration chromatographies; (c) the exhibit identical thermostability; and (d) the phosphotyrosyl-protein phosphatase activity is sensitive to inhibition by p-nitrophenyl phosphate and by several classical inhibitors of prostatic acid phosphatase including l(+)-tartrate, molybdate, vanadate and NaF. The purified enzyme exhibits high specificity towards phosphotyrosyl-proteins with little activity towards several phosphoseryl-proteins and phosphothreonyl-proteins examined. The present findings indicate that prostatic acid phosphatase may function in vivo as a phosphotyrosyl-protein phosphatase.

Fetal bovine bone cells synthesize bone-specific matrix proteins.

Abstract: We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.

Macrophage-derived growth factor for osteoblast-like cells and chondrocytes

Abstract: Rat resident peritoneal macrophages in primary culture were found to elaborate a mitogenic factor (or factors) for rat osteoblast-like cells and chondrocytes but not for skin fibroblasts. Growth-promoting activity appeared in the incubation medium within the first 20 hr of macrophage culture and was released in amounts that paralleled the number of macrophages per culture. After their proliferative response, as judged by increases in DNA synthesis and cell number, the osteoblast-like cells became enriched in alkaline phosphatase, an index of osteoblast specialization. The macrophage-derived activity was nondialyzable and heat-stable, and it was eliminated by exposure to trypsin. Inhibition of prostaglandin cyclooxygenase failed to modify its generation. Partial purification (Amicon filter concentration, gel filtration) disclosed principal peaks of activity corresponding to Mr of 43,000 and 10,000. The crude conditioned medium and the Mr 43,000-peak, but not the low-molecular-weight peak, exhibited interleukin 1 activity, as judged by the ability to stimulate the proliferation of mouse thymic lymphocytes. The macrophage-derived growth factor described herein may participate in bone remodeling and repair and in primary bone and cartilage growth.

Preparation and characterization of monoclonal antibodies against placental alkaline phosphatase and other human trophoblast-associated determinants.

Abstract: We have prepared monoclonal antibodies by immunizing BALB/c mice with purified human term placental plasma membranes. The antibodies were selected to show predominant specificity for trophoblast and trophoblast derivatives. Four of these antibodies have been found to recognize the placenta-specific isozyme of alkaline phosphatase (EC 3.1.3.1), and to cross-react with the closely related testis form of this enzyme. One antibody recognized transferrin, a serum protein with an abundant placental receptor. The specificities of the other antibodies, whose target antigens are unknown, are described. Their reactivity with some human tumour-derived epithelial cell lines suggests that they may provide useful markers of human trophectoderm differentiation, as well as for properties selected for during tumour progression.

The effects of prostaglandin E2, parathyroid hormone, 1,25 dihydroxycholecalciferol, and cyclic nucleotide analogs on alkaline phosphatase activity in osteoblastic cells

Abstract: Clone MC3T3-E1 cells isolated from newborn mouse calvaria had the same type of alkaline phosphatase (ALP) as that found in adult mouse calvaria (the liver-bone-kidney type), as judged by polyacrylamide gel electrophoresis as well as by heat lability and amino acid inactivation. The effects of prostaglandin E2 (PGE2), parathyroid hormone (PTH), 1,25 dihydroxycholecalciferol [1,25(OH)2D3], and adenosine-3′, 5′-cyclic monophosphate (cAMP) analogs on ALP were investigated. PGE2 and 1,25(OH)2D3 increased ALP activity in dose-related manner with a maximal effect at concentrations of 10 ng/ml and 40 pg/ml, respectively. N6,O2-dibutyryl adenosine-3′, 5′-cyclic monophosphate (DBcAMP) also induced an increase in ALP activity in a dose-related fashion with a maximal effect at a concentration of 0.5 mM which was 2.2-fold over that of the controls. Induced ALP was of the “liver-bone-kidney” type. Antinomycin D and cycloheximide inhibited the increase in ALP activity induced by DBcAMP. The level of ALP was elevated by 8-bromo-adenosine-3′,5′-cyclic monophosphate and theophylline, but not by N6,O2-dibutyryl guanosine-3′,5′-cyclic monophosphate and sodium butyrate. Moreover, PGE2 dramatically increased the level of cAMP in the cells with a maximal effect at a concentration of 10 ng/ml, indicating that PGE2 and DBcAMP induce an increase of ALP activity in clone MC3T3-E1 cells by increasing the cAMP level; PTH did not affect enzyme activity and cAMP, level in the cells. These results suggest that PGE2, DBcAMP, and 1,25(OH)2D3 are involved in bone formationin vivo as well.

Effect of dietary supplements of guar gum and cellulose on intestinal cell proliferation, enzyme levels and sugar transport in the rat

Abstract: Male Wistar rats (approximately 200 g) were given fibre-free semi-synthetic diets containing either sucrose (S) or a sucrose-starch mixture (SS) as the carbohydrate component, or a diet similar to SS containing 40 g guar gum/kg (G), or 100 g cellulose/kg (C). The animals remained healthy, and weight gain after 30 d was similar in all groups. The small intestines of the animals given diet G were significantly longer than those of the other groups, and showed signs of increased mitotic activity and mucosal growth. No significant differences in mucosal enzyme activity were detected between the two fibre-free control groups. Lactase (EC 3.2.1.23) and alkaline phosphatase (EC 3.1.3.1) activities were significantly lower than controls in group G, but were higher in group C. Kinetic analysis of 3-O-methyl glucose uptake by isolated intestine indicated that the maximum transport rate (Vmax) of tissue from group G tended to be lower than from the fibre-free group SS and group C. It is concluded that materials which are classed as dietary fibre but which differ markedly in their physical properties may also differ in the functional changes to which they give rise in the small intestine. These changes may be at least partially mediated by effects on mucosal cell proliferation.

Arachidonic acid metabolism in a cell suspension isolated from rabbit renal outer medulla

Abstract: We studied arachidonic acid (AA) metabolism by a cell suspension containing principally cells of the thick ascending limb of the loop of Henle (TALH) obtained from the inner stripe of the outer medulla of the rabbit kidney. Based on comparison of specific activities of enzymes before and after separation, alkaline phosphatase, Na+-K+-adenosine triphosphatase, as well as Tamm-Horsfall glycoprotein and electron microscopic appearance, 80% of these cells were estimated to be TALH in origin. TALH cells had low activity of cyclooxygenase and did not show evidence of lipoxygenase activity. However, they selectively converted exogenous AA to oxygenated metabolites by a cytochrome P-450 related mechanism. AA metabolites were produced in large amounts (30-40% conversion of [14C]AA, 1 to 5 micrograms/mg of protein/30 min) and were increased 5-fold after separation of TALH cells from a suspension of outer medullary cells, suggesting that TALH cells synthesized these metabolites. Induction of cytochrome P-450 by pretreatment of rabbits with beta-naphthoflavone and 3-methylcholanthrene increased formation of the AA metabolites by almost 2-fold in the separated cells and correlated with cytochrome P-450 content of the renal outer medulla. Additionally, SKF 525A and carbon monoxide inhibited product formation in these renomedullary cells, supporting a role for a cytochrome P-450-like monooxygenase in TALH cell function.

Serum gamma glutamyl transferase as a specific indicator of bile duct lesions in the rat liver.

Abstract: Serum gamma-glutamyl transferase (GGT), a marker of hepatic injury used extensively in humans, has been used rarely in rats because its specificity has not been previously defined. Studies were designed for investigation of the specificity of serum GGT activity with the use of cell type specific hepatotoxicants in Fischer 344 rats. Single necrogenic doses of CCl4, allyl alcohol (AA), and alpha-naphthylisothiocyanate (ANIT) were used to produce cell specific injury in centrilobular hepatocytes, periportal hepatocytes, and bile duct cells, respectively. Administration of CCl4 markedly increased serum activities of alanine aminotransferase (ALT), alkaline phosphatase (AP), and serum bile acid concentrations within 24 hours but had no effect on serum GGT activity. ANIT treatment increased serum GGT and AP activities and bile acid concentration 24 hours following administration. Allyl alcohol administration increased serum ALT activity but had no effect on GGT activity. Administration of ANIT in the diet at 0.01%, 0.022%, 0.047%, and 0.1% for 2, 4, and 6 weeks produced dose- and time-dependent increases in serum GGT activity which strongly correlated with quantitative increases in hepatic bile duct volume, which was determined morphometrically. These observations support the use of serum GGT activity in the rat as diagnostic of bile duct cell necrosis when increases are detected shortly after the insult and as an indicator of possible bile duct hyperplasia.

Phosphorylation in vivo of red‐muscle pyruvate kinase from the channelled whelk, Busycotypus canaliculatum, in response to anoxic stress

Abstract: That red muscle pyruvate kinase from anoxic Busycotypus canaliculatum (PK-anoxic) is a phosphoprotein was demonstrated by the anoxia-dependent, in vivo, covalent incorporation of injected [32P]orthophosphate into the enzyme molecule. Specificity in labelling of PK-anoxic was strongly suggested by: (a) coincidental elution of pyruvate kinase activity and radioactivity following chromatography of purified PK-anoxic on Sepharose CL-6B, and (b) comigration of the area containing [32P]phosphate and Coomassie-Blue-staining protein following SDS-polyacrylamide gel electrophoresis of homogenous PK-anoxic. The [32P]phosphate content of the enzyme was calculated to be 7.3 mol phosphate/mol enzyme (233 kDa, 180 units/mg protein). Evidence for the reversibility of this phosphorylation was provided by the consistent kinetic similarities between purified red muscle pyruvate kinase from aerobic animals (PK-aerobic) and homogenous, unlabelled, alkaline phosphatase treated PK-anoxic. Comparison of the electrophoretic mobilities of products derived from acid hydrolysis of purified 32P-labelled PK-anoxic with authentic substances suggest the presence of an O-phospho-L-threonine residue in the protein. That this residue plays a probable role in an interconversion mechanism was suggested by the lack of phosphate exchange of homogenous 32P-labelled PK-anoxic in the presence of all substrates. A possible role of protein phosphorylation as a mechanism for the overall control of molluscan anaerobic metabolism is suggested.

Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles.

Abstract: In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA). Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energized membrane and not simply from ATP. Treatment of the vesicles with protease, under conditions that did not interfere with subsequent protein synthesis, also inactivated them for subsequent translocation. We conclude that export of some proteins requires protein-containing machinery in the cytoplasmic membrane that derives energy from the proton motive force. Images

Inhibition of human natural killer cell reactivity by exogenous adenosine 5'-triphosphate.

Abstract: The presence of micromolar concentrations (5 to 100 microM) of adenosine 5'-triphosphate (ATP) in cytotoxicity assays of human natural killer (NK) cells with K562 targets resulted in marked inhibition of NK activity NK activity of peripheral blood mononuclear cells (PBL) and of purified NK cells (ie, large granular lymphocytes (LGL] were similarly sensitive to inhibition by ATP The inhibitory activity was specific to ATP and was not demonstrated with GTP, UTP, CTP, or with other adenosine compounds This inhibitory activity resulted from an effect of ATP on the effector cells and was not dependent on serum components ATP did not inhibit binding of the LGL to target cells, and therefore the inhibition by ATP is presumed to be related to some post-recognition metabolic events Some physiologic role in regulation of NK activity by ATP seems possible, because nonspecific phosphatases (bacterial alkaline phosphatase or human alkaline phosphatase) stimulated NK activity and partially reversed the ATP-induced inhibition of reactivity

Effect of aminoglycoside antibiotics on cellular functions of kidney epithelial cell line (LLC-PK1): A model system for aminoglycoside nephrotoxicity

Abstract: The effects of aminoglycoside antibiotics on cellular functions of the LLC-PK1 kidney epithelial cell line were studied as a model system for aminoglycoside nephrotoxicity. The treatment with aminoglycoside antibiotics for 3 days caused a decrease in the dome number in the confluent LLC-PK1 cells and an increase in the floating cells in the culture medium. The inhibition of dome formation was dose-dependent and the rank-order of the degree of inhibition was compatible with the rank-order of in vivo nephrotoxicity. Aminoglycosides also decreased the intracellular content of cyclic AMP, with a correlation between the alteration of dome formation and cyclic AMP content. The specific activities of N-acetyl-beta-D-glucosaminidase (marker for lysosomes), aminopeptidase and alkaline phosphatase (marker for apical membranes) and (Na++K+)-adenosine triphosphatase (marker for basolateral membranes) in the homogenate were decreased by gentamicin treatment. Lysosomal and apical membrane enzymes released into the culture medium were increased by gentamicin treatment. The ultrastructural alterations in the lysosomes of gentamicin-treated cells also were observed. Above results suggest that aminoglycoside toxicity to LLC-PK1 cells may be similar to that reported for renal tubules.